Chronic hepatitis C virus (HCV) infection is a common cause of serious liver diseases, including cirrhosis and hepatocellular carcinoma. Approximately 2% of the world's population is infected with this virus, rendering it a major health threat. In the U.S., treatment with a combination of interferon-alpha and ribavirin, the only currently licensed therapies for this disease, eliminates the virus from less than 50% of those infected with the most prevalent HCV genotype, genotype 1. HCV NS3 encodes several important enzyme activities required for RNA replication, including protease, helicase and NTPase. NS3 as the protease is essential for viral replication as it proteolytically processes viral polyprotein, which is required for viral replicase complex assembly, and thus an important target in antiviral drug development efforts against HCV infection. The goal of this project is to understand the molecular mechanism of action of NS3 protein in HCV RNA replication in cultured Huh7 cells, by examining HCV replicon mutants, which have shown significant differences in transient replication levels, with a few amino acid changes within the protease domain of the NS3. The aims of this project are: Specific aim1: To determine if replication-enhancing mutations in NS3 alter HCV nonstructural protein processing and to determine whether altered nonstructural protein processing results in differential RNA replication; Specific aim 2: To determine if NS3 modulates RNA replication through interaction with NS5A. The outcome of this application will enhance basic molecular biological knowledge concerning viral RNA replication, improve the currently available system to study HCV, and help future drug development efforts leading to enhanced therapy against this dangerous pathogen.